Phosphorus-31 nuclear magnetic resonance spectroscopy (31P-NMR) was used to monitor changes in the levels of several high-energy compounds both prior to and following the initiation of motility in spermatozoa of the horseshoe crab, Limulus polyphemus L. These spermatozoa are easily collected and are nonmotile until stimulated with a sperm-motility-initiating peptide (SMI) which can be extracted from Limulus eggs. Following the addition of SMI, several changes in metabolite levels within the spermatozoa were obtained, the most significant of which were a decrease in nucleotide triphosphate (NTP) and phosphoarginine levels and an increase in the levels of inorganic phosphate (Pi), NDP, and phosphomonoesters. An increase in the intracellular pH (0.45 pH units) of the sperm was also noted following the completion of SMI-stimulated motility.Since the landmark 31P nuclear magnetic resonance spectroscopy (NMR) studies of erythrocytes by Moon and Richards ('73) and of rat muscle by Hoult et al. ('74), the use of NMR, a noninvasive analytical tool, has continued to influence the biological sciences. In addition to the nondisruptive aspect of NMR, the major strengths of this technique have been in the measurement of metabolite concentrations (Burt et al.. '76) and intracellular pH (Gillies et al., '82). ' Spermatozoa are ideal for 31P-NMR studies since large quantities of free cells can be obtained and examined either in their storage environment (as semen) or in a selected environment. It is thus surprising that so few studies have been performed on semen or spermatozoa; 31P-NMR studies of semen were first performed on rooster semen (Burt and Chalovich, '78) The semen of Limulus polyphemus is considered a model system for 31P-NMR examination of metabolism, since 1) spermatozoa can be observed in a resting stage prior to motility (Brown, '76; Clapper and Brown, '80a); 2) motility can be initiated through the addition of a small peptide, sperm motility initiator (SMI), which is extracted from the surface of Limulus eggs (Clapper and Epel, '82a,b; Barnum and Brown, '83); and 3) initiation of the acrosome reaction can be triggered in a systematic fashion through the use of SMI in Kf-free artificial seawater (Clapper and Epel, '85). Thus, Limulus semen permits the 31P-NMR observation of spermatozoa at three distinct stages: at rest, following motility, and after the acrosome reaction. It should be noted that spermatozoa of other species do not enable the investigator to examine separately these three stages (Clapper and Epel, '82a). In addition, it has been well established that Limulus spermatozoa can be rendered motile by using metal 0 1986 ALAN R. LISS, INC.
